Previous results from our laboratory established that the N-terminal 70% of NS2A and the C-terminal 8 amino acids of NS1 are required for NS1-NS2A cleavage. To further delineate the requirements for NS1-NS2A cleavage, we constructed recombinant vaccinia viruses expressing chimeric proteins consisting of PreM fused to the 3 or 8 C-terminal amino acids of NS1 plus NS2A. The results showed that all of NS1, with the exception of the 8 amino acid cleavage site, is dispensable for NS1-NS2A cleavage. Also, a signal sequence is required for cleavage to occur as the chimeric protein Sig PreM-NS1(8)-NS2A was not cleaved. This suggests that targeting to the exocytic pathway is a prerequisite for NS1-NS2A cleavage. Additional experiments were performed to analyze this cleavage. Brefeldin A, a drug which blocks transport out of the Golgi, did not block NS1/NS2A cleavage. This suggests that NS1/NS2A cleavage occurs in the Golgi or in a compartment between the ER and the Golgi. NS1/NS2A cleavage was studied during a chase under conditions that block ER-to-Golgi transport: incubation with the drug carbonyl cyanide m-chlorophenylhydrazone (CCCP), or incubation at 14oC. Both of these treatments completely blocked the chase of uncleaved NS1-NS2A precursor into NS1 product. This is consistent with the notion that NS1/NS2A cleavage occurs after exit from the ER. Further elucidation of the cleavage mechanism is now possible using an in vitro ER-to-Golgi transport system recently developed by others.